Fructosamine reagent and calibrator system

Abstract

A system for determining the concentration of fructosamine in sera which consists of a first reagent in which a tetrazolium salt which reduces all reactive substances in sera including fructosamine and a second reagent which is responsive to all reactive substance in sera other than fructosamine. The difference in color change as between the two allows the determination of concentration of fructosamine.

What is claimed is: 
     
       1. A reagent system for the determination of fructosamine in sera which comprises: (a) a first alkaline aqueous reagent containing a tetrazolium salt which reacts with all reactive substances in sera including fructosamine and interfering substances to exhibit a measurable first change of color within a predetermined time span of less than about 10 minutes in consequence of the reaction of the tetrazolium salt with fructosamine and the interfering substances; and   (b) a second reagent identical to the first alkaline aqueous reagent and boric acid in an amount sufficient to inhibit reaction of fructosamine with the tetrazolium salt to produce by reduction of the tetrazolium salt with the reactive substances including interfering substances other than fructosamine a measurable second change of color in the same predetermined time span as the first reagent to enable the determination of fructosamine concentration in sera by the difference in the first color change produced using the first alkaline aqueous reagent which reacts with fructosamine and the second color change produced using the second alkaline aqueous reagent each color change produced on the same proportional amount of sera and after identical predetermined time spans.   
     
     
       2. The reagent system as claimed in claim 1 in combination with a liquid standard containing a predetermined amount of fructosamine which when used with said first and second alkaline aqueous reagents enable the determination of a factor used as multiplier used to compute from the difference in color change the fructosamine concentration in a sera sample. 
     
     
       3. An assay system as claimed in claim 2 further including a liquid normal level fructosamine control and a liquid high level fructosamine control. 
     
     
       4. An assay system as claimed in claim 3 further including a liquid normal fructosamine level control having a fructosamine range of 229-281 μmol, when assayed, and a liquid high fructosamine level control having a fructosamine range, when assayed, of 457-559 μmol. 
     
     
       5. An assay system as claimed in claim 2 further including a liquid normal level fructosamine control having a fructosamine range of 229-281 μmol, when assayed, and a liquid high fructosamine level control having a fructosamine range, when assayed, of 457-559 μmol. 
     
     
       6. A reagent system for the determined of fructosamine in sera at 37° C. which comprises: (a) a first liquid reagent which is an aqueous solution of a carbonate buffer present in a concentration from about 0.1 to about 0.4 molar, a tetrazolium salt which is reduced by fructosamine and interfering substances in sera present in a concentration of about 0.1 to about 5 millimolar urea in a concentration of about 0.1 to about 1 molar, octylphenoxy polyethoxyethanol in a concentration of about 0.1 to about 4% volume by volume, and from 0 to 3% v/v ethoxylated nonylphenol, said solution having a pH of from about 8.5 to about 11.5; and   (b) a second liquid reagent identical to the first reagent and further containing boric acid in a concentration of about 0.05 to about 1 molar, said first reagent producing a first color within a predetermined time span of less than 10 minutes and said second reagent producing a second color during the same time span to enable by color difference a determination of fructosamine in sera.   
     
     
       7. A reagent system as claimed in claim 6 in which the tetrazolium salt is p-Iodonitrotetrazolium Violet. 
     
     
       8. A reagent system as claimed in claim 7 in combination with a standard containing a known amount of fructosamine which when used with said first and second liquid alkaline aqueous reagents to determine a factor used as a multiplier to determine from the difference in color change fructosamine concentration in a sera sample. 
     
     
       9. A reagent system as claimed in claim 6 in combination with a standard containing a known amount of fructosamine which when used with said first and second liquid alkaline aqueous reagents to determine a factor used as a multiplier to determine from the difference in color change fructosamine concentration in a sera sample. 
     
     
       10. A reagent system for the determination of fructosamine in sera which comprises: (a) a first liquid reagent containing 0.5 millimolar p-Iodonitrotetrazolium Violet, about 0.1 molar sodium carbonate monohydrate, about 0.1 molar sodium bicarbonate, about 0.1 molar urea, about 1% volume by volume of octylphenoxy polyethoxyethanol, and 1% volume by volume ethoxylated nonylphenol dissolved in the deionized water and having a pH of about 11; and   (b) a second liquid reagent identical to the first reagent but further including about 0.12 molar boric acid and also having a pH of about 11, said reagent system being stable for at least 1 year at 2°-10° C. and cooperating with the first liquid reagent to enable completion of a fructosamine assay determined by a difference in color change using the first liquid reagent and the second liquid reagent in the same proportional amount of sera during identical periods of time, both within 10 minutes at 37° C.   
     
     
       11. A reagent system as claimed in claim 7 in combination with a liquid standard containing a predetermined amount of fructosamine which when used with said first and second liquid alkaline aqueous reagents enable the determination a factor used as a multiplier used to compute from the difference in color change produced by the first and second liquid reagents the fructosamine concentration in a sera sample. 
     
     
       12. A reagent system as claimed in claim 11 in which the standard provides a fructosamine value in the range of about 300 to 500 μmol. 
     
     
       13. A method for determining fructosamine in sera which comprises: (a) determining a first absorbance of a first color produced by reaction of a first liquid reagent containing a tetrazolium salt with a first amount of sera, said first color produced in consequence of reduction by fructosamine and other interfering substances in said sera of said tetrazolium salt, said first absorbance determined at two predetermined points in time, each within 10 minutes at 37° C.;   (b) determining a second absorbance of a second color produced by reaction of a second liquid reagent in an amount equal to the amount of said first liquid reagent with sera in an amount equal to said first amount of said sera using a second liquid reagent containing a tetrazolium salt and boric acid which produces a second color change by reaction of the tetrazolium salt with interfering substances in the sera other than fructosamine, said boric acid being in an amount sufficient to inhibit the reaction of fructosamine with the tetrazolium salt, said second absorbance being determined at two predetermined points in time, each within 10 minutes at 37° C.; and   (c) determining the fructosamine concentration in said sera as a function of the difference in the first and second color change produced using the first liquid reagent which reacts with fructosamine and the second liquid reagent which is inhibited from reacting with fructosamine.   
     
     
       14. A method of determining the concentrate of fructosamine in sera at 37° C. which comprises: (a) adding to a first sample of sera a first liquid reagent which reacts with fructosamine and interfering substances and is an aqueous solution of a carbonate buffer present in a concentration of from about 0.1 to about 0.4 molar, a tetrazolium salt which is reduced by fructosamine and interfering substances in sera present in a concentration of about 0.1 to about 1 molar of a 100% by volume octylphenoxy polyethoxyethanol in a concentration of about 0.1 to about 4% v/v and from 0 to 3% v/v ethoxylated nonylphenol, said solution having a pH of from about 8.5 to about 11.5 and measuring the change in absorbance of the sera added reagent at 500 nm at 2 or 3 minutes of the addition of the reagent and at 2 minutes after the first measurement of absorbance;   (b) repeating the procedure of step (a) using a second sample of the sera of the same volume as the first sample of sera by addition of a second liquid reagent identical in volume and content of the first reagent and further containing boric acid in a concentration of about 0.05 to about 1 molar to inhibit reaction with fructosamine; and   (c) determining from the difference in change of absorbance in step (a) and the change of absorbance in step (b) the fructosamine concentration in the sera.   
     
     
       15. A method as claimed in claim 14 in which the tetrazolium salt is p-Iodonitrotetrazolium violet. 
     
     
       16. A method of determining the concentrate of fructosamine in sera which comprises: (a) adding to a predetermined amount of sera a first liquid reagent containing 0.5 millimolar p-Iodonitrotetrazolium Violet, about 0.1 molar sodium carbonate monohydrate, about 0.1 molar sodium bicarbonate, about 0.1 molar urea, about 1% v/v octylphenoxy polyethoxyethanol, and 1% w/v ethoxylated nonylphenol dissolved in the deionized water and having a pH of about 11 and determining absorbance at 37° C. of 500 nm 2 and 4 minutes or 3 and 5 minutes after addition of the first liquid reagent to the sera;   (b) repeating step (a) by adding to another sample of the sera, of same proportional amount as the first, a second liquid reagent identical in proportional volume and content of the first reagent but further containing about 0.12 molar boric acid and having a pH of about 11; and   (c) using the difference in absorbance determined in step (a) and the differential in absorbance determined in step (b) to determine the fructosamine content of the sera.